Premature aging and immune senescence

in HIV-infected children
Ketty Gianesina, Antoni Noguera-Julianb, Marisa Zanchettac,
Paola Del Biancoc, Maria Raffaella Petraraa, Riccardo Fregujaa,
Osvalda Rampond, Clàudia Fortunyb, Mireia Camóse, Elena Mozzod,
Carlo Giaquintod and Anita De Rossia,c

Objective: Several pieces of evidence indicate that HIV-infected adults undergo

premature aging. The effect of HIV and antiretroviral therapy (ART) exposure on the
aging process of HIV-infected children may be more deleterious since their immune
system coevolves from birth with HIV.
Design: Seventy-one HIV-infected (HIVþ), 65 HIV-exposed-uninfected (HEU), and 56
HIV-unexposed-uninfected (HUU) children, all aged 0–5 years, were studied for
biological aging and immune senescence.
Methods: Telomere length and T-cell receptor rearrangement excision circle levels
were quantified in peripheral blood cells by real-time PCR. CD4þ and CD8þ cells were
analysed for differentiation, senescence, and activation/exhaustion markers by flow
cytometry.
Results: Telomere lengths were significantly shorter in HIVþ than in HEU and HUU
children (overall, P < 0.001 adjusted for age); HIVþ ART-naive (42%) children had
shorter telomere length compared with children on ART (P ¼ 0.003 adjusted for age).
T-cell receptor rearrangement excision circle levels and CD8þ recent thymic emigrant
cells (CD45RAþCD31þ) were significantly lower in the HIVþ than in control groups
(overall, P ¼ 0.025 and P ¼ 0.005, respectively). Percentages of senescent
(CD28CD57þ), activated (CD38þHLA-DRþ), and exhausted (PD1þ) CD8þ cells were
significantly higher in HIVþ than in HEU and HUU children (P ¼ 0.004, P < 0.001, and
P < 0.001, respectively). Within the CD4þ cell subset, the percentage of senescent cells
did not differ between HIVþ and controls, but programmed cell death receptor-1
expression was upregulated in the former.
Conclusions: HIV-infected children exhibit premature biological aging with acceler-
ated immune senescence, which particularly affects the CD8þ cell subset. HIV infection
per se seems to influence the aging process, rather than exposure to ART for prophylaxis
or treatment. Copyright ß 2016 Wolters Kluwer Health, Inc. All rights reserved.

AIDS 2016, 30:1363–1373

Keywords: immune activation, immune senescence, microbial translocation,

pediatric HIV/AIDS, premature aging, telomere length, T-cell receptor
rearrangement excision circle

a
Department of Surgery, Oncology and Gastroenterology – DiSCOG, Section of Oncology and Immunology, Unit of Viral
Oncology and AIDS Reference Center, University of Padova, Padova, Italy, bUnitat d’Infectologia, Servei de Pediatria; Hospital
Sant Joan de Déu, Universitat de Barcelona, Barcelona, Spain, cIstituto Oncologico Veneto (IOV) – IRCCS, dDepartment of Mother
and Child Health, University of Padova, Padova, Italy, and eClinical Laboratory Department, Hematology Unit, Hospital San Joan
de Déu, Universitat de Barcelona, Barcelona, Spain.
Correspondence to Professor Anita De Rossi, Section of Oncology and Immunology, Unit of Viral Oncology and AIDS Reference
Center, Department of Surgery, Oncology and Gastroenterology – DiSCOG, University of Padova, Via Gattamelata 64, 35128
Padova, Italy.
Tel: +39 049 8215894; fax: +39 049 8072854; e-mail: anita.derossi@unipd.it
Received: 6 October 2015; revised: 25 February 2016; accepted: 7 March 2016.
DOI:10.1097/QAD.0000000000001093
ISSN 0269-9370 Copyright Q 2016 Wolters Kluwer Health, Inc. All rights reserved. This is an open-access article distributed under the terms
of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License, where it is permissible to download and share the work
provided it is properly cited. The work cannot be changed in any way or used commercially. 1363
1364 AIDS 2016, Vol 30 No 9
Introduction
The introduction of antiretroviral therapy (ART) has
changed the natural history of pediatric HIV infection;
ART-based prophylaxis regimens have in fact reduced
mother-to-child transmission of HIV from 15–20% to
under 2% in high-income countries, and have also given
rise to substantial improvements in terms of survival and
quality of life in HIV-infected children [1,2]. HIV
infection is, therefore, now considered a chronic disease
which persists for many decades [3]. However, despite
improvements in immune function and reduction of
AIDS-related complications, including opportunistic
infections and AIDS-associated malignancies, ART does
not restore full health. Many studies have demonstrated
that ART-treated HIV-infected adults have a higher risk
of non-AIDS-related overall morbidity and mortality
compared with age-matched HIV-uninfected individuals.
This increased risk is mainly because of a range of non-
AIDS-defining illnesses associated with aging, including
malignancies [4–8], and it has been advanced that the
increase in non-AIDS-defining diseases among HIV-
infected patients may be because of premature aging [9].
The pathogenic mechanism underlying this increased risk
is still poorly understood. Chronic immune activation
because of the persistence of circulating HIV virions may
play a key role in the senescent pathway. Activated cells
undergo clonal expansion in response to viral persistence,
resulting in differentiation and accumulation of non-
functional senescent cells [10]. It has been also advanced
that premature and accelerated aging in HIV-infected
patients can be because of adverse effects of antiretroviral
drugs. Nucleoside reverse transcriptase inhibitors have
been shown to inhibit telomerase activity in replicating
cell lines in vitro, leading to accelerated shortening of
telomere length [11,12].
The clinical complications of HIV infection and ART
treatment in children may be more serious than in adults.
The course of vertically transmitted infection in infants is
characterized by faster disease progression and shorter
time to AIDS, compared with adults. After infection,
plasma HIV-RNA levels are higher in infants than in
adults. In addition, they persist at high levels and decline
slowly with age in the absence of ART [13,14], whereas
in adults control of viral load is reached a few weeks after
infection [15]. This slower control of viral replication is
probably because of the fact that the immune system is still
maturing. The innate immunity in children is of
particular importance and plays a critical role in HIV
pathogenesis [16–18], as the adaptive immune system is
still developing [19]. HIV infection since birth together
with long-term exposure to ART may affect premature
aging and immune senescence in HIV-infected children
even more than in adults.
To date, few data are available on premature aging in
HIV-infected children [20–23] and no reports give a
comprehensive assessment of telomere shortening, a key
molecular marker of biological aging, together with the
activation/senescent profile of T lymphocytes. In this
study, we analysed biological aging in relation to immune
activation and senescence markers in a cohort of
perinatally HIV-infected children.
Methods
Ethic statement
The study was approved by the Ethics Committees of the
Azienda Ospedaliera Padova (Prot. n.#2921P) and the
Hospital Sant Joan de Déu, Universitat de Barcelona
(Prot. n.#04–15); written informed consent was
obtained for all children from their parents/guardians.
Study population
A total of 71 perinatally HIV-infected (HIVþ), 65 HIV-
exposed-uninfected (HEU), and 56 HIV-unexposed-
uninfected (HUU) children, all aged 0–5 years, were
included in this study. HIVþ and HEU children attended
the Department of Mother and Child Health, University
of Padova or the Infectious Diseases Unit, Pediatrics
Department, Hospital Sant Joan de Déu, Universitat de
Barcelona. For each HIVþ and HEU child, the first
cryopreserved sample available after birth was chosen for
the study. None of the HIVþ or HEU children was
breastfed. HUU children were recruited at Pediatric
Emergency Department of Azienda Ospedaliera Padova
or Hospital Sant Joan de Déu, Universitat de Barcelona.
Exclusion criteria were malignancies, chronic infections,
sarcoidosis, diabetes mellitus type-1, rheumatoid arthritis,
and systemic lupus erythematosus.
Sample preparation
Peripheral blood mononuclear cells (PBMC) were
isolated from ethylenediaminetetraacetic acid-treated
peripheral blood (2–5 ml) by centrifugation on a
Ficoll-Paque gradient (Pharmacia, Uppsala, Sweden).
PBMC were cryopreserved and plasma samples were
stored in liquid nitrogen and at 808C, until use.
Telomere length measurement by quantitative
real-time PCR
Relative telomere length was determined by mono-
chrome quantitative multiplex PCR assay [24] with
minor modifications. Each PCR reaction was performed
in a final volume of 25 ml containing 5 ml sample
(2 ng DNA/ml) and 20 ml reaction mix containing
0.75  SYBR GreenI (Invitrogen, Italy), 10 mmol/l
Tris-hydrochloric acid pH 8.3, 50 mmol/l potassium
chloride, 3 mmol/l magnesium chloride, 0.2 mmol/l
each deoxynucleotide (dNTP) (Applied Biosystems,
Foster City, California, USA), 1 mmol/l dithiothreitol,
0.625 U AmpliTaq Gold DNA polymerase, 1% dimethyl
sulfoxide (Sigma-Aldrich, St Louis, Missouri, USA), and

900 nmol/l of each of the primers. Telomere and
albumin gene primers sequences are described in [24].
PCR reactions were performed on a LightCycler480
real-time PCR detection system (Roche Applied
Science, Mannheim, Germany). The thermal cycling
profile was 15 min at 958C, two cycles of 15 s at 948C, 15 s
at 498C, followed by 40 cycles of 15 s at 948C, 10 s
at 628C, 15 s at 748C, 10 s at 848C, 15 s at 888C, with
signal acquisition at the end of both the 748C and 888C
steps. A standard curve was generated at each PCR run,
consisting of DNA from the RAJI cell line, serially
diluted from 100 to 0.41 ng/ml [25]. LightCycler raw
text files were converted using the LC480Conversion
free software (http://www.hartfaalcentrum.nl/index.
php?main¼files&fileName¼ LC480Conversion.zip&de
scription¼LC480%20Conversion&sub¼LC480Convers
ion), and the data were analysed using LinRegPCR free
software [26]. All samples were blindly and consecutively
run in triplicate together with reference samples. The
intra and interassay reproducibility of both telomere and
albumin PCR results was evaluated using dilutions of the
reference curve. Telomere length values were calculated
as telomere/single-copy gene (T/S) ratio, as previously
described [25]. The intra and interassay variability of
T/S values was evaluated using reference samples;
coefficients of variation were 3.98% or less and 8.14%
or less, respectively.
T-cell receptor rearrangement excision circle
levels quantification
Thymic output in PBMC was studied by measurement of
T-cell receptor rearrangement excision circle (TREC)
levels by real-time PCR, as previously described [27].
TREC levels were expressed as the number of TREC
copies/105 PBMC.
Viral load quantification
Plasma HIV-RNA levels were determined in all HIV-
infected children using the COBAS Taqman HIV-1 test
(Roche, Branchburg, New Jersey, USA). The lower limit
of detection was 50 HIV-RNA copies/ml. HIV-DNA
levels in PBMC were measured by real-time PCR, and
expressed as HIV-DNA copies/106 PBMC as previously
described [27].
Flow cytometry analysis
T-cell phenotyping was performed on cryopreserved
PBMC. Cells were thawed, washed, stained for 20 min in
the dark with the Live/Dead Fixable Near-IR Dead
Cell Stain Kit (Life Technologies, Carlsbad, California,
USA) and with fluorescent-conjugated mononuclear
antibodies CD3-fluorescein isothiocyanate, CD31-
phycoerythrin (PE), CD38-PE, CD57-PE, CD45RA-
allophycocyanin (APC) and programmed cell death
receptor (PD)-1-PECy7 (Becton-Dickinson, San Diego,
California, USA), CD27-PECy7 (Beckman Coulter,
Fullerton, California, USA) and CD4-VioBlue,
CD8-VioGreen, Human Leukocyte Antigen - antigen
Pediatric HIV infection and aging Gianesin et al. 1365
D Related (HLA-DR)-APC, and CD28-APC (Miltenyi
Biotec, Auburn, California, USA). Appropriate isotypic
controls (mouse IgG1-PE, IgG2b-APC, and IgG1k-
PECy7) were used to evaluate nonspecific staining. Cells
were then washed and resuspended in phosphate-
buffered saline supplemented with 1% paraformalde-
hyde. All samples were analysed using LSRII Flow
cytometer (Becton-Dickinson). A total of 100 000 events
were collected in the lymphocyte gate using morpho-
logical parameters (forward and side-scatter). Data were
processed with FACSDivaSoftware (Becton-Dickinson)
and analysed using KaluzaAnalyzing Software v.1.2
(Beckman Coulter) (Supplementary Figure 1, http://
links.lww.com/QAD/A903). Samples for flow-cytome-
try analysis were available for 24 HIVþ children, 21
HEU, and 18 HUU children. The characteristics of
these subgroups are given in Supplementary Table 1,
http://links.lww.com/QAD/A904.
Quantification of soluble markers
Plasma levels of soluble CD14 (sCD14), IL-6, and TNFa
were quantified with commercially available assays
(Human sCD14, IL-6, and TNFa Quantikine ELI-SA;
R&D Systems, Minneapolis, Minnesota, USA) according
to the manufacturer’s protocol. Samples for analysis of
soluble markers were available for 24 HIVþ, 21 HEU, and
18 HUU children.
Statistical analysis
Unadjusted comparisons of continuous variable distri-
butions among groups were assessed with the Kruskal–
Wallis nonparametric test, and the associations
between categorical variables were analysed by the
x2 test. Spearman’s r coefficient (rs) was used for
correlations. Normal distributions for telomere
length and TREC levels were visually checked by
quantile–quantile plots. Linear regression models esti-
mated the telomere length and TREC levels in HIVþ,
HEU, and HUU children, ART exposure and naive
groups, log-transformed HIV-RNA and HIV-DNA
covariates, adjusted for age and its interaction with
groups. Samples with undetectable plasma HIV-RNA
were assigned a value of 20 copies/ml to include them in
the statistical analyses. All statistical analyses were
performed with Statistical Analysis Software (SAS)
(Release 9.2; SAS Institute, Cary, North Carolina,
USA). Adjustments for multiple testing with Hochberg’s
correction were made for comparisons of CD4þ and
CD8þ cell subsets among groups. All P values were two-
tailed, and were considered significant at less than 0.05.
Results
Characteristics of study population
The characteristics of the study population are listed in
Table 1. The median age of HIVþ children was 3.11
1366 AIDS 2016, Vol 30 No 9

[interquartile range (IQR) 1.41–4.48], 1.73 (0.99–3.20)
for HEU, and 1.85 (0.85–3.46) years for HUU children.
Thirty of the 71 (42%) HIVþ children were ART-naive,
the others were on ART [median time 18 (11–36.5)
months]. ART-naive HIVþ children were younger than
those on therapy [1.70 (0.69–3.59) and 3.62 (2.17–4.81)
years, respectively; P ¼ 0.004]. Sixty-one of the 65
(93.8%) HEU children had been exposed to ART
prophylaxis during gestation [median 34 (17–38) weeks]
and received postnatal prophylaxis [6 weeks of zidovudine
(ZDV) monotherapy]. Of the 61 ART-treated pregnant
women, 40 (65.6%) had received triple therapy based on
ZDV þ lamivudine (3TC) þ nevirapine (NVP), 17
(27.8%) ZDV þ 3TC þ protease inhibitor, and the
remaining 4 (6.6%) a regimen based on tenofovir
(TDF) þ emtricitabine (FTC) þ protease inhibitor.
Telomere length is shorter in HIV-infected
children than in controls
The median telomere length value in PBMC was
significantly lower in HIVþ than in HEU and HUU
children, being 2.21 (1.94–2.58), 2.63 (2.25–3.21), and
2.88 (2.49–3.1), respectively [P < 0.001 not age adjusted
(Fig. 1a); P < 0.001 age adjusted]. Telomere length values
significantly decreased with age in HEU and HUU
(regression coefficient (b) ¼ 0.0102, P ¼ 0.008 and
b ¼ 0.0100, P ¼ 0.011, respectively), but not in HIVþ
children (b ¼ 0.0018, P ¼ 0.587) (Fig. 1b). Of note, in
the HIVþ group, ART-naive children had shorter
telomere length compared with those on ART [2.11
(1.75–2.37) ‘vs.’ 2.46 (2.07–2.68); P ¼ 0.006 not age
adjusted (Fig. 1c); P ¼ 0.003 age adjusted]. Telomere
lengths were not associated with age in either ART-naive
(b ¼ 0.0054, P ¼ 0.227) or ART-treated children
(b ¼ 0.0046, P ¼ 0.258) (Fig. 1d). After adjusting for
age, mean telomere length values tended to decrease with
increasing HIV-RNA levels (b ¼ 0.0396; P ¼ 0.056),
but not with HIV-DNA levels (b ¼ 0.0004, P ¼ 0.490),
and were significantly lower in ART-naive children
(b ¼ 0.3913, P ¼ 0.003). In the final multivariate
model, including all variables, the stepwise method
selected only ART exposure as a significant predictor
variable of higher mean telomere length value
(P ¼ 0.039).
Thymic output is lower in HIV-infected children
than in controls
HEU and HUU children had higher TREC levels than
HIVþ children [5409 (3470–6600), 5370 (2380–8102),
3498 (2051–6780)] TREC copies/105 PBMC, respec-
tively [P ¼ 0.018 not age adjusted (Fig. 1e); P ¼ 0.025
age-adjusted]. TREC levels decreased significantly with
increasing age in HEU and HUU groups (b ¼ 61,
P ¼ 0.009 and b ¼ 86, P ¼ 0.001, respectively), but not
in HIVþ children (b ¼ 17, P ¼ 0.353) (Fig. 1f). No
significant differences in TREC dynamics were found
between ART-treated and ART-naive children (Fig. 1g
and h).
Phenotypic T-cell alterations occur early in HIV-
infected children
There were no differences in the frequencies of CD3þ
cells among the three groups (P ¼ 0.590) (Table 2). The
percentages of total CD4þ cells were lower in the HIVþ
than in the control groups (P ¼ 0.001). Within CD4þ
cells, HIVþand control groups did not significantly differ
in percentages of naive (CD45RAþCD27þ), central
memory (CD45RACD27þ), effector memory
(CD45RACD27), and terminally differentiated
(CD45RAþCD27) cell subsets (Table 2). However,
when central and effector memory cell subsets, the major
cellular reservoirs for HIV [28], were considered
together, they tended to be lower in HIVþ than in
HEU and HUU children [21.9 (15.3–38.3)% ‘vs’ 29.8

Table 1. Demographic and clinical characteristics of HIVR, HIV-exposed-uninfected children, and HIV-unexposed-uninfected children.

HIVþ (n ¼ 71) HEU (n ¼ 65) HUU (n ¼ 56)

Age, median (IQR) years 3.11 (1.40–4.48) 1.74 (0.99–3.31) 1.85 (0.84–3.46)
Sex, n (%)
Men 39 (55%) 34 (52%) 29 (52%)
Ethnicity/race, n (%)
White 49 (69%) 47 (72.3%) 49 (87.5%)
Black 20 (28.2%) 15 (23.1%) 5 (9.0%)
Asian 2 (2.8%) 3 (4.6%) 2 (3.5%)
Exposed to ART prophylaxis, n (%) 5 (7%) 61 (93.8%) –
Exposed to ART, n (%) 41 (58%) – –
Duration of ART exposure, median (IQR) months 18 (12–36) – –
Percentage of lifetime on ART 57.5 (42.6–84.5) – –
Detectable plasmaviremia at sample collection, n (%) 54/71 (76.1%) – –
ART naive 30/30 (100%) – –
On ART 17/41 (58.5%)
Plasmaviremia at sample collection (log10 copies/ml)
ART naive 5.31 (4.90–5.62) – –
On ART 3.96 (2.70–5.27) – –

ART, antiretroviral therapy; HEU, HIV-exposed-uninfected children; HIVþ, HIV-infected children; HUU, HIV-unexposed-uninfected children;
IQR, interquartile range.
 Pediatric HIV infection and aging Gianesin et al. 1367

(a) (b)
5.0 5.0
P < 0.001

Telomere length (T/S ratio)

Telomere length (T/S ratio)

4.0 4.0

3.0 3.0

2.0 2.0

1.0 1.0
HIV+ β=–0.0018, P = 0.587
HEU β=–0.0102, P = 0.008
HUU β=–0.0100, P = 0.011
0 0
HIV+ HEU HUU 0 20 40 60 80
Age (months)
(c) (d)
5.0 4.0
P = 0.006
Telomere length (T/S ratio)

Telomere length (T/S ratio)

4.0
3.0

3.0
2.0
2.0

1.0
1.0
on ART β=–0.0046, P = 0.258
0
ART-naive β=–0.0054, P = 0.227
0
ART-naive on ART 0 20 40 60 80
Age (months)
(e) (f)
20000 20000
P = 0 .018 HIV+ β=–17, P = 0.353
HEU β=–64, P = 0.009
TREC copies/105 PBMC

TREC copies/105 PBMC

HUU β=–86, P = 0.001

15000 15000

10000 10000

5000 5000

0 0
HIV+ HEU HUU 0 20 40 60 80
(g) Age (months)
(h)
15000 14000
on ART β=–8, P = 0.754
P = 0.300 ART-naive β=–11, P = 0.680
12000
TREC copies/105 PBMC

TREC copies/105 PBMC

10000
10000
8000

6000
5000
4000

2000

0 0

ART-naive on ART 0 20 40 60 80
Age (months)

Fig. 1. HIV-infected (HIVR) children have shorter telomere length (TL) and lower T-cell receptor rearrangement excision circle
(TREC) levels than HIV-exposed-uninfected (HEU) and HIV-unexposed-uninfected (HUU) children. (a) TL values not age-
adjusted in HIVþ (n ¼ 71), HEU (n ¼ 65), and HUU (n ¼ 56) children. (b) TL as function of age in HIVþ (black circles, continuous
line), in HEU (gray squares, continuous line), and HUU (white circles, dotted line) children. (c) TL values not age-adjusted in
HIVþ children, subdivided into antiretroviral therapy (ART)-naive (n ¼ 30) and on ART (n ¼ 41). (d) TL as function of age in HIVþ
children, subdivided into ART-naive (gray squares) and ART-treated (black circles). (e) TREC levels not age-adjusted in HIVþ
(n ¼ 71), HEU (n ¼ 65), and HUU (n ¼ 56) children. (f) TREC levels as function of age in HIVþ (black circles, continuous line), in
HEU (gray squares, continuous line) and HUU (white circles, dotted line) children. (g) TREC levels not age-adjusted among HIVþ
children, subdivided into ART-naive (n ¼ 30) and on ART (n ¼ 41). (h) TREC levels as function of age in HIVþ children, subdivided
into ART-naive (gray squares) and on ART (black circles). Boxes and whiskers: 25–75th and 10–90th percentiles, respectively;
central line in boxes: median. b, regression coefficient.
1368 AIDS 2016, Vol 30 No 9

Table 2. Frequencies of CD4R and CD8R T-cell subsets.

HIVþ (n ¼ 24) % median (IQR) HEU (n ¼ 21) % median (IQR) HUU (n ¼ 18) % median (IQR) Overall P value

CD3þ 64.4 (58.8–69.2) 61.8 (49.8–68.4) 60.2 (54.6–66.4) 0.590

CD4þ 39.6 (35.1–45.3) 53.4 (43.5–64.7) 52.5 (38.0–60.6) 0.001
Naive 76.8 (59.9–84.4) 70.1 (59.9–81.0) 64.7 (56.9–75.3) 0.423
Central memory 18.1 (13.2–31.1) 27.3 (17.4–32.6) 27.9 (20.7–35.1) 0.206
Effector memory 3.4 (1.2–5.8) 2.5 (1.8–6.3) 4.4 (2.9–7.8) 0.220
T. differentiated 0.5 (0.2–1.0) 0.3 (0.1–0.6) 0.4 (0.3–0.1) 0.330
RTE 63.6 (54.9–72.4) 58.3 (46.0–68.2) 54.2 (49.3–61.7) 0.181
PEC 12.4 (5.3–16.4) 11.7 (8.2–16.1) 10.4 (8.1–15.2) 0.738
Senescent 0.4 (0.1–0.7) 0.2 (0.1–0.5) 0.2 (0.1–1.3) 0.568
Activated 3.3 (2.2–5.9) 2.1 (1.3–3.5) 2.8 (1.6–3.8) 0.041
Exhausted 4.1 (3.4–6.6) 3.5 (1.9–5.2) 3.2 (2.7–5.1) 0.050
CD8þ 31.2 (25.8–39.6) 28.9 (21.2–35.0) 25.9 (22.8–29.0) 0.063
Naive 46.2 (37.6–75.1) 77.1 (55.9–84.7) 71.0 (46.7–86.1) 0.019
Central memory 11.0 (6.9–23.2) 16.3 (10.9–26.5) 12.7 (9.8–18.9) 0.278
Effector memory 7.1 (2.3–13.1) 2.1 (1.1–4.9) 4.3 (1.3–11.4) 0.033
T. differentiated 16.3 (4.5–36.4) 2.5 (1.0–8.6) 4.2 (2.1–16.2) 0.001
RTE 55.3 (41.4–71.8) 69.8 (60.4–80.1) 68.1 (59.5–79.3) 0.005
PEC 17.1 (6.5–29.2) 5.9 (3.8–15.3) 9.8 (4.6–15.2) 0.040
Senescent 25.8 (12.4–43.2) 8.5 (6.8–16.7) 9.7 (3.3–27.3) 0.004
Activated 7.0 (5.2–12.2) 4.7 (3.9–7.6) 3.4 (2.8–6.7) <0.001
Exhausted 7.1 (5.0–12.4) 3.5 (2.1–5.9) 3.7 (2.4–5.3) <0.001

HEU, HIV-exposed-uninfected children; HIVþ, HIV-infected children; HUU, HIV-unexposed-uninfected children; IQR, interquartile range; PEC,
peripheral expanded cells; RTE, recent thymic emigrant cells.

(18.6–39.4)% and 35.6 (26.2–42.6)%; P ¼ 0.085]. The
percentages of senescent cells (CD4þCD28CD57þ)
were similar in the three groups, whereas activated
CD38þHLA-DRþ and exhausted PD-1þ were more
expanded in HIVþ children than in controls (Table 2). In
particular, within the HIVþ group, the percentages of
CD4þCD38þHLA-DRþ and CD4þPD-1þ were higher
in children with detectable viral load than in aviremic
children (P ¼ 0.056 and P ¼ 0.037, respectively).
The median of CD8þ cell percentages tended to be
higher in HIVþ children than in control groups
(P ¼ 0.063). Notably, significant differences emerged
among CD8þ T-cell subsets. HIVþ children showed
lower percentage of naive cells than HEU and HUU
children [46.2 (37.6–75.1)%, 77.1 (55.9–84.7)%, 71.0
(46.7–86.1)%; P ¼ 0.019]. In particular, HIVþ children
had a lower frequency of CD8þ recent thymic emigrant
[recent thymic emigrant cells (RTE), CD45RAþCD31þ)
cells (P ¼ 0.005) and a higher percentage of peripheral
expanded cells [peripheral expanded cells (PEC)
CD45RAþCD31] than control groups (P ¼ 0.040),
suggesting strong peripheral cell proliferation (Table 2). In
addition, the percentage of CD8þ RTE cells decreased
with age in HEU and HUU but not in HIVþ children
(Fig. 2a-c ). Interestingly, CD8þ RTE cells were lower in
children with detectable viral load than in aviremic
children [41.8 (22.6–64.4)% ‘vs.’ 55.9 (53.6–76.7)%;
P ¼ 0.039] (not shown); plasma HIV-RNA tended to be
inversely correlated with CD8þ RTE cells (rs ¼ 0.363,
P ¼ 0.080) and positively correlated with CD8þ PEC
(rs ¼ 0.357, P ¼ 0.085) (Fig. 2d).
Among memory cell subsets, the frequency of CD8þ
central memory did not differ among HIVþ, HEU, and
HUU children (P ¼ 0.278). However, in the HIVþ
group, this subset was more expanded in children with
detectable viremia than in those with undetectable HIV-
RNA (P ¼ 0.027) (not shown). Both effector memory
(CD45RACD27) and terminally differentiated cells
(CD45RAþCD27) were more expanded in HIVþ
children than in control groups (Table 2). In HIVþ
children, the proportion of senescent CD8þ cells was also
higher than in HEU and HUU groups [25.8 (12.4–
43.2)% ‘vs.’ 8.5 (6.8–16.7)% and 9.7 (3.3–27.3)%;
P ¼ 0.004]. This expansion was particularly observed in
children with detectable plasmaviremia, indicating that
active HIV replication stimulates the production of a
senescent phenotype [41.6 (18.5–45.8)% ‘vs.’ 4.4 (2.1–
13.4)%; P ¼ 0.002]. In addition, the activation of CD8þ
cells was significantly higher in HIVþ children than in
controls [7.0 (5.2–12.2)% ‘vs.’ 4.7 (3.9–7.6)% in HEU
and 3.4 (2.8–6.7)% in HUU; P < 0.001] (Table 2), and
significantly increased with HIV-RNA levels (rs ¼ 0.672;
P < 0.001) (not shown). The percentages of CD8þPD-
1þ cells were significantly higher in HIVþ than in HEU
and HUU children (P ¼ 0.003 and P < 0.001, respect-
ively) (Table 2). After adjustments for multiple testing, the
CD4þ cell subset, and the CD8þ terminally differen-
tiated, activated, exhausted, senescent, and RTE cell
subsets remained significantly different (P < 0.05). Also,
the levels of proinflammatory cytokines IL-6 and TNFa
associated with the senescent phenotype [29–31] were
higher in HIVþ than in HEU and HUU children
(Supplementary Table 2, http://links.lww.com/QAD/
A905), thus supporting the accelerated immune senes-
cence in HIVþ children. PD-1 expression in viremic
subjects was also significantly higher than in those with
undetectable plasmaviremia (P ¼ 0.002) and was corre-
lated with HIV-RNA levels (rs ¼ 0.471, P ¼ 0.021) and
 Pediatric HIV infection and aging Gianesin et al. 1369

(a) (b)
100 100

80 80
% CD8+ RTE cells

% CD8+ RTE cells

60 60

40 40

20 20
rs = −0.523 rs = −0.486
P = 0.025 P = 0.026
0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Age (months) Age (months)
(c) (d)
100 100 100
CD8+ RTE CD8+ PEC
rs = −0.363, P = 0.080 rs = 0.357, P = 0.085
80 80 80

% CD8+ PEC cells

% CD8+ RTE cells
% CD8+ RTE cells

60 60 60

40 40 40

20 20 20
rs = −0.242
P = 0.252
0 0 0
0 10 20 30 40 50 60 70 1 2 3 4 5 6
log10 HIV-RNA (copies/mL)
Age (months)

Fig. 2. Relationship between recent thymic emigrant (RTE) CD8R cells with age and HIV plasmaviremia. Percentage of CD8þ
RTE cells as function of age in (a) HUU, (b) HEU, and (c) HIVþ children. (d) Levels of HIV plasmaviremia in relation to percentages
of CD8þ RTE (black circles, continuous line) and CD8þ peripheral expanded cells (PEC) (white circles, dotted line) among HIVþ
children. rs, Spearman’s r correlation coefficient.

CD8þCD38þHLA-DRþ cells (rs ¼ 0.528, P ¼ 0.009) Discussion

(not shown).
This is the first study describing biological aging and
Telomere length values were inversely correlated with immune senescence in HIV-infected children compared
percentages of senescent (Fig. 3a), activated (Fig. 3d) and with HIV-exposed-uninfected and unexposed-unin-
exhausted CD8þ cells (Fig. 3g) in HIVþ, but not in HEU fected children, all aged 0–5 years. Overall, the results
(Fig. 3b, e, h) or HUU children (Fig. 3c, f, i). demonstrate that HIV-infected children exhibit prema-
ture biological aging with accelerated immune senes-
cence which affects the CD8þ T-cell subset in particular.
HIV-infected children have increased gut
microbial translocation In contrast to the study of Côté et al. [21], which found no
Median levels of sCD14 were significantly higher in difference in telomere length between HIVþ children
HIVþ than in HEU and HUU children [2699 (2333– and controls, in our study telomere length was
2826) ‘vs.’ 2138 (1954–2427) and 2065 (1850–2480) significantly shorter in HIVþ than in HEU and HUU
ng/ml; P < 0.001] (Supplementary Figure 2A, http:// children. This discordant result may be because of the
links.lww.com/QAD/A903). In HIVþ children, sCD14 different ages of the two cohorts: the children enrolled in
levels positively correlated with HIV-RNA levels our study were younger (aged 0–5 years, median 3.1)
(rs ¼ 0.585, P ¼ 0.007), with percentages of CD8þHLA- than those of Côté et al. (aged 0–19 years, median 13.3).
DRþCD38þ (rs ¼ 0.418, P ¼ 0.065), and CD8þPD-1þ As telomere shortening in peripheral blood cells is very
(rs ¼ 0.645, P ¼ 0.002), but not with percentages of rapid during the first years of life [32], the difference
CD8þCD28CD57þ (rs ¼ 0.172, P ¼ 0.487) or telomere between HIV-infected children and controls may have
length (rs ¼ 0.386, P ¼ 0.124) (Supplementary Figure emerged more clearly in our cohort. The two cohorts also
2b-f, http://links.lww.com/QAD/A903). differed in duration of ART exposure. The longer
1370 AIDS 2016, Vol 30 No 9

(a) (b) (c)

HIV+ HEU HUU
5.0 5.0 5.0
Telomere length (T/S ratio)

Telomere length (T/S ratio)

Telomere length (T/S ratio)

rs = −0.458 rs = −0.036 rs = −0.186
4.0 P = 0.054 4.0 P = 0.882 4.0 P = 0.498

3.0 3.0 3.0

2.0 2.0 2.0

1.0 1.0 1.0

0 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 0 10 20 30 40
(d) % senescent CD8+ cells (e) % senescent CD8+ cells (f) % senescent CD8+ cells

5.0
Telomere length (T/S ratio)

5.0 5.0

Telomere length (T/S ratio)

Telomere length (T/S ratio)

rs = −0.515 rs = −0.026 rs = -0.110
4.0 P = 0.019 4.0 P = 0.911 4.0 P = 0.656

3.0 3.0 3.0

2.0 2.0 2.0

1.0 1.0 1.0

0 0 0
0 5 10 15 20 25 30 0 5 10 15 20 0 5 10 15
% activated CD8+ cells % activated CD8+ cells % activated CD8+ cells
(g) (h) (i)
5.0 5.0 5.0
Telomere length (T/S ratio)

Telomere length (T/S ratio)

Telomere length (T/S ratio)

rs = −0.637 rs = −0.112 rs = −0.018
4.0 P = 0.002 4.0 P = 0.641 4.0 P = 0.934

3.0 3.0 3.0

2.0 2.0 2.0

1.0 1.0 1.0

0 0 0
0 5 10 15 20 25 30 0 5 10 15 0 5 4 6 8 10
% exhausted CD8+ cells % exhausted CD8+ cells % exhausted CD8+ cells

Fig. 3. Relationship between telomere length (TL) and T-cell immunophenotypic profiles. TL values in relation to percentages of
senescent (CD8þCD28CD57þ) cells in (a) HIVþ, (b) HEU, and (c) HUU children, activated (CD8þCD38þHLA-DRþ) cells in (d)
HIVþ, (e) HEU, and (f) HUU children, and exhausted (CD8þPD-1þ) cells in (g) HIVþ, (h) HEU, and (i) HUU children. HIVþ, HIV-
infected; HEU, HIV-exposed uninfected; HUU, HIV-unexposed uninfected; rs, Spearman’s r correlation coefficient.

exposure to ART of children in the above study (median significantly shorter telomeres than the latter. All these
85 months of ART exposure) may explain the loss of findings indicate that HIV infection per se, rather than
association found in our study population, consisting of exposure to therapy, influences the aging process. Also,
ART-naı̈ve children or ones recently on ART (median 18 the finding that over 90% of HEU children had been
months of ART exposure). perinatally exposed to ARTand that their telomere length
did not differ from those of HUU children indicates that
Nucleoside reverse transcriptase inhibitors are known relatively short-term ARTexposure does not significantly
inhibitors of telomerase reverse transcriptase, and have influence telomere length, matching the results of a
been reported to be associated with telomere shortening recent report [36]. It is possible that longer exposure to
[11,12]. The inverse association between telomere length ART in HIV-infected children exacerbates HIV-driven
and ART-exposure (median time 123 months) has telomere shortening.
recently been demonstrated in a cohort of adults, but
the small sample size and lack of optimal control of ART- The mechanism behind telomere shortening in HIV-
naive patients preclude definite conclusions [33]. Two infected individuals is unknown. Short telomeres may be
other studies of HIV-infected adults [34,35] confirm the because of excessive cellular replication after chronic
association of telomere length shortening with HIV immune activation, but the virus itself may play an active
status, but not with relatively short ART exposure role. Telomerase activity is in fact severely impaired in
(median times 58 and 48 months, respectively). uninfected CD34þ hematopoietic progenitor cells iso-
lated from HIV-infected patients [37]. HIV infection and
In our group of HIV-infected children, the ART-naive HIV-Tat protein also downmodulate telomerase expres-
ones, even though younger than those on ART, had sion and activity in lymphoblastoid cells [38] and

peripheral blood lymphocytes [39–41]. Although the
CD4þ T cell is the target of HIV infection, we found
that CD8þ T-cell compartment was largely impaired in
the HIV-infected children. They had a lower frequency
of CD8þ naive cells than controls and a decline in this
cell subset did not correlate with age, as occurs in HIV-
uninfected children. In particular, the decreased
percentages of RTE cells with increased percentages
of PEC together with increasing levels of HIV
plasmaviremia indicate that HIV induces peripheral
proliferation of CD8þ cells and their differentiation
into effector cells, which play a central role in
immunity against pathogens [42]. As already described
in adults and older children [22,43,44], the decrease in
naive cell subset is associated with skewed maturation of
CD8þ cells toward an effector phenotype which,
without adequate replenishment of new CD8þ naive
cells, induces accumulation of cells with a senescent
phenotype. A major driver of the cellular senescent
phenotype is telomere shortening [29]. Our data
indicate that HIV-infected children accumulate
CD8þCD38þ and CD8þPD-1þ cells together with a
higher percentage of senescent CD8þ cells. The finding
that activated and exhausted CD8þ cells are inversely
correlated with telomere length supports the idea that
persistent immune activation and cellular exhaustion
are closely linked to accelerated biological aging and
immune senescence. Chronic viral coinfections may
induce immune activation and accelerate immune
senescence [35,45,46]. In particular, it has been shown
that cytomegalovirus (CMV) leads to significant
changes in the CD8þ repertoire [45,46]. Unfortunately,
clinical data on CMV serology were available only for
HIVþ and a subgroup of HEU children: 40 of 71
(56.3%) HIVþ and nine of 33 (27.3%) HEU children
were CMV-positive. The CMVþ and CMV sub-
groups of HIV-infected children did not significantly
differ as regards telomere length and markers of
immunological profile. Larger studies are needed to
better understand the contribution of coinfections in
the aging process of HIV-infected children.
The finding that sCD14 levels were correlated with
percentages of activated/exhausted CD8þ cells and the
lack of correlation between sCD14 and senescent CD8þ
cells and/or telomere length suggest that the gut damage
and gastrointestinal lymphocyte depletion which occurs
during HIV infection do not directly cause immune
senescence, but they do contribute to accelerated aging
and senescence through immune system activation.
Overall, our data support a scenario in which viremia
leads to high turnover with continual loss and output of
naı̈ve cells, which rapidly differentiate and exhaust their
effector function, resulting in an accumulation of
senescent cells with short telomeres. These data are
consistent with a previous report [47] showing that high
percentage of CD8þCD28 correlates with shorter
telomeres.
Pediatric HIV infection and aging Gianesin et al. 1371
The evidence that immune senescence is more serious in
children with detectable HIV viremia focuses attention
on the need for early and long-standing control of HIV
replication and chronic immune activation. In particular,
this aspect must be considered when a treatment
interruption has been planned [48]. As previously
demonstrated in a pediatric cohort, viremia does impair
the immune reconstitution of memory and effector
CD4þ T-cell subsets [49], and also contributes to the
expansion of T-regulatory cells [50] which may influence
the specific HIV immune response, allowing the virus to
replicate and increase immune activation.
In conclusion, HIV-infected children exhibit premature
biological aging with accelerated immune senescence,
which particularly affects the CD8þ T-cell subset. The
shorter telomere length and higher percentage of
senescent cells in HIVþ children, compared with
children on ART or HEU, suggest that HIV infection
per se, rather than exposure to ART for prophylaxis or
treatment, influences the aging process. These data
support the importance of maintaining undetectable viral
load to avoid premature immune senescence and
dysfunction of CD8þ cells, compromising their tumor
immune surveillance function and increasing the risk of
age-related diseases, including malignancies.
Acknowledgements
We are grateful to Camino Estella and Francesco
Carmona for their skillful technical assistance in the
Hospital Sant Joan de Deu, Barcelona, and in the Unit of
Viral Oncology and AIDS Reference Center, Depart-
ment of Surgery, Oncology and Gastroenterology.
This work was supported by the Paediatric European
Network for Treatment of AIDS (PENTA) Foundation,
and from the European Union Seventh Framework
Programme (FP7/2007–2013) under grant agreement
n8 260694.
Authors’ contributions: K.G. designed and performed the
experiments, undertook clinical data collection, data
analysis and interpretation, wrote the first draft, carried
out critical revision and provided intellectual input to
further drafts. A.N.J. and C.F. provided clinical samples
and clinical data, carried out critical revision and provided
intellectual input to further drafts. O.R., E.M., and M.C.
provided clinical samples and clinical data. P.D.B.
provided statistical expertise and data analysis. Z.M.,
M.R.P., and R.F. performed the experiments. C.G.
carried out critical revision and provided intellectual
input to further drafts. A.D.R. conceived and designed
the study, carried out data analysis and interpretation,
undertook critical revision and provided intellectual
input to further drafts. All the authors approved the final
version of this article.
1372 AIDS 2016, Vol 30 No 9
Conflicts of interest
There are no conflicts of interest.
Partial presentation at: 22nd Conference on Retroviruses and
Opportunistic Infections, Seattle, Washington, USA, 23–26
February 2015.
References
1. Gibb DM, Duong T, Tookey PA, Sharland M, Tudor-Williams G,
Novelli V, et al. National Study of HIV in Pregnancy and
Childhood Collaborative HIV Paediatric Study. Decline in
mortality, AIDS, and hospital admissions in perinatally HIV-
1 infected children in the United Kingdom and Ireland. BMJ
2003; 327:1019.
2. Judd A, Doerholt K, Tookey PA, Sharland M, Riordan A, Menson
E, et al., Collaborative HIV Paediatric Study (CHIPS); National
Study of HIV in Pregnancy and Childhood (NSHPC). Morbidity,
mortality, and response to treatment by children in the United
Kingdom and Ireland with perinatally acquired HIV infection
during 1996-2006: planning for teenage and adult care. Clin
Infect Dis 2007; 45:918–924.
3. Deeks SG, Lewin RS, Havlir DV. The end of AIDS: HIV infection
as a chronic disease. Lancet 2013; 382:1525–1533.
4. Powles T, Robinson D, Stebbing J, Shamash J, Nelson M,
Gazzard B, et al. Highly active antiretroviral therapy and the
incidence of non-AIDS-defining cancers in people with HIV
infection. JCO 2008; 27:884–890.
5. Crum-Cianflone N, Hullsiek KH, Marconi V, Weintrob A, Ga-
nesan A, Barthel RV, et al. Trends in the incidence of cancers
among HIV-infected persons and the impact of antiretroviral
therapy: a 20-year cohort study. AIDS 2009; 23:41–50.
6. Simard EP, Pfeiffer RM, Engels EA. Cumulative incidence of
cancer among people with AIDS in the United States. Cancer
2011; 117:1089–1096.
7. Guaraldi G, Orlando G, Zona S, Menozzi M, Carli F, Garlassi E,
et al. Premature age-related comorbidities among HIV-infected
persons compared with the general population. Clin Infect Dis
2011; 53:1120–1126.
8. Chiappini E, Berti E, Gianesin K, Petrara MR, Galli L, Giaquinto
C, et al. Pediatric human immunodeficiency virus infection and
cancer in the highly active antiretroviral treatment (HAART)
era. Cancer Lett 2014; 347:38–45.
9. Effros RB, Fletcher CV, Gebo K, Halter JB, Hazzard WR, Horne
FM, et al. Aging and infectious diseases: workshop on HIV
infection and aging: what is known and future research direc-
tions. Clin Infect Dis 2008; 47:542–553.
10. Desai S, Landay A. Early immune senescence in HIV disease.
Curr HIV/AIDS Rep 2010; 7:4–10.
11. Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizush-
ina Y, et al. 3’-Azido-2’,3’-dideoxynucleoside 5’-triphosphates
inhibit telomerase activity in vitro, and the corresponding
nucleosides cause telomere shortening in human HL60 cells.
Nucleic Acids Res 2007; 35:7140–7149.
12. Hukezalie KR, Thumati NR, Côté HC, Wong JM. In vitro and ex
vivo inhibition of human telomerase by anti-HIV nucleoside
reverse transcriptase inhibitors (NRTIs) but not by non-NRTIs.
PLoS One 2012; 7:e47505.
13. De Rossi A, Masiero S, Giaquinto C, Ruga E, Comar M, Giacca
M, et al. Dynamics of viral replication in infants with vertically
acquired human immunodeficiency virus type 1 infection.
J Clin Invest 1996; 97:323–330.
14. McIntosh K, Shevitz A, Zaknun D, Kornegay J, Chatis P, Karthas
N, et al. Age- and time-related changes in extracellular viral
load in children vertically infected by human immuno-
deficiency virus. Pediatr Infect Dis J 1996; 15:1087–1091.
15. Henrard DR, Phillips JF, Muenz LR, Blattner WA, Wiesner D,
Eyster ME, et al. Natural history of HIV-1 cell-free viremia.
JAMA 1995; 274:554–558.
16. Ricci E, Malacrida S, Zanchetta M, Montagna M, Giaquinto C,
De Rossi A. Role of beta-defensin-1 polymorphisms in mother-
to-child transmission of HIV-1. J Acquir Immune Defic Syndr
2009; 51:13–19.
17. Freguja R, Gianesin K, Zanchetta M, De Rossi A. Cross-talk
between virus and host innate immunity in pediatric HIV-1
infection and disease progression. New Microbiol 2012;
35:249–257.
18. Gianesin K, Freguja R, Carmona F, Zanchetta M, Del Bianco P,
Malacrida S, et al. The role of genetic variants of stromal cell-
derived factor 1 in pediatric HIV-1 infection and disease
progression. PLoS One 2012; 7:e44460.
19. Prendergast AJ, Klenerman P, Goulder PJR. The impact of
differential antiviral immunity in children and adults. Nat
Rev Immunol 2012; 12:636–648.
20. Mansoor N, Abel B, Scriba TJ, Hughes J, de Kock M, Tameris M,
et al. Significantly skewed memory CD8R T cell subsets in
HIV-1 infected infants during the first year of life. Clin Immunol
2009; 130:280–289.
21. Côté HC, Soudeyns H, Thorne A, Alimenti A, Lamarre V, Maan
EJ, et al., the CIHR Emerging Team in HIV therapy, aging
(CARMA). Leukocyte telomere length in HIV-infected and
HIV-exposed uninfected children: shorter telomeres with un-
controlled HIV viremia. PLoS One 2012; 7:e39266.
22. Dı́az L, Méndez-Lagares G, Correa-Rocha R, Pacheco YM,
Ferrando-Martı́nez S, Ruiz-Mateos E, et al. Detectable viral
load aggravates immunosenescence features of CD8 T-cell
subsets in vertically HIV-infected children. J Acquir Immune
Defic Syndr 2012; 60:447–454.
23. Méndez-Lagares G, Dı́az L, Correa-Rocha R, León Leal JA,
Ferrando-Martı́nez S, Ruiz-Mateos E, et al. Specific patterns
of CD4-associated immunosenescence in vertically HIV-
infected subjects. Clin Microbiol Infect 2013; 19:558–565.
24. Cawthon RM. Telomere length measurement by a novel mono-
chrome multiplex quantitative PCR method. Nucleic Acids Res
2009; 37:e21.
25. Rampazzo E, Bertorelle R, Serra L, Terrin L, Candiotto C,
Pucciarelli S, et al. Relationship between telomere shortening,
genetic instability, and site of tumour origin in colorectal
cancers. Br J Cancer 2010; 102:1300–1305.
26. Ruijter JM, Ramakers C, Hoogars WMH, Karlen Y, Bakker O,
van den Hoff MJB, et al. Amplification efficiency: linking base-
line and bias in the analysis of quantitative PCR data. Nucleic
Acids Res 2009; 37:e45.
27. Ometto L, De Forni D, Patiri F, Trouplin V, Mammano F,
Giacomet V, et al. Immune reconstitution in HIV-1-infected
children on antiretroviral therapy: role of thymic output and
viral fitness. AIDS 2002; 16:839–849.
28. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA,
Yassine-Diab B, et al. HIV reservoir size and persistence are
driven by T cell survival and homeostatic proliferation. Nat
Med 2009; 15:893–900.
29. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad
things happen to good cells. Nat Rev Mol Cell Biol 2007; 8:729–
740.
30. Kuilman T, Peeper DS. Senescence-messaging secretome: SMS-
ing cellular stress. Nat Rev Cancer 2009; 9:81–94.
31. Young AR, Narita M. SASP reflects senescence. EMBO Rep
2009; 10:228–230.
32. Zeichner SL, Palumbo P, Feng Y, Xiao X, Gee D, Sleasman J,
et al. Rapid telomere shortening in children. Blood 1999;
93:2824–2830.
33. Leeansyah E, Cameron PU, Solomon A, Tennakoon S, Velayud-
ham P, Gouillou M, et al. Inhibition of telomerase activity by
human immunodeficiency virus (HIV) nucleos(t)ide reverse
transcriptase inhibitors: a potential factor contributing to
HIV-associated accelerated aging. J Infect Dis 2013;
207:1157–1165.
34. Pathai S, Lawn SD, Gilbert CE, McGuinness D, McGlynn L,
Weiss HA, et al. Accelerated biological ageing in HIV-infected
individuals in South Africa: a case-control study. AIDS 2013;
27:2375–2384.
35. Zanet DL, Thorne A, Singer J, Maan EJ, Sattha B, Le Campion A,
et al. Association between short leukocyte telomere length and
HIV infection in a cohort study: no evidence of a relationship
with antiretroviral therapy. Clin Infect Dis 2014; 58:1322–
1332.
36. Imam T, Jitratkosol MHJ, Soudeyns H, Sattha B, Gadawski I,
Maan E, et al. Leukocyte telomere length in HIV-infected
pregnant women treated with antiretroviral drugs during preg-
nancy and their uninfected infants. J Acquir Immune Defic
Syndr 2012; 60:495–502.

37. Vignoli M, Stecca B, Furlini G, Re MC, Mantovani V, Zauli G,
et al. Impaired telomerase activity in uninfected haematopoie-
tic progenitors in HIV-1-infected patients. AIDS 1998; 12:999–
1005.
38. Reynoso R, Minces L, Salomon H, Quarleri J. HIV-1 infection
downregulates nuclear telomerase activity on lymphoblastoic
cells without affecting the enzymatic components at the tran-
scriptional level. AIDS Res Hum Retrov 2006; 22:425–429.
39. Ballon G, Ometto L, Righetti E, Cattelan AM, Masiero S,
Zanchetta, et al. Human immunodeficiency virus type 1 mod-
ulates telomerase activity in peripheral blood lymphocytes.
J Infect Dis 2001; 183:417–424.
40. Franzese O, Adamo R, Pollicita M, Comandini A, Laudisi A,
Perno CF, et al. Telomerase activity, hTERT expression, and
phosphorylation are downregulated in CD4(R) T lymphocytes
infected with human immunodeficiency virus type 1 (HIV-1).
J Med Virol 2007; 79:639–646.
41. Comandini A, Naro C, Adamo R, Akbar AN, Lanna A,
Bonmassar E, et al. Molecular mechanisms involved in HIV-
1-Tat mediated inhibition of telomerase activity in human CD4
(R) T lymphocytes. Mol Immunol 2013; 54:181–192.
42. Northfield JW, Loo CP, Barbour JD, Spotts G, Hecht FM,
Klenerman P, et al. Human immunodeficiency virus type 1
(HIV-1)-specific CD8R T(EMRA) cells in early infection are
linked to control of HIV-1 viremia and predict the subsequent
viral load set point. J Virol 2007; 81:5759–5765.
43. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ,
Crotty LE, et al. Expression of CD57 defines replicative senes-
cence and antigen-induced apoptotic death of CD8R T cells.
Blood 2003; 101:2711–2720.
Pediatric HIV infection and aging Gianesin et al. 1373
44. Montesano C, Anselmi A, Palma P, Bernardi S, Cicconi R, Mattei
M, et al. HIV replication leads to skewed maturation of
CD8-positive T-cell responses in infected children. New Micro-
biol 2010; 33:303–309.
45. Pawelec G, Derhovanessian E. Role of CMV in immune senes-
cence. Virus Res 2011; 157:175–179.
46. Solana R, Tarazona R, Aiello AE, Akbar AN, Appay V, Beswick
M, et al. CMV and Immunosenescence: from basics to clinics.
Immun Ageing 2012; 9:23.
47. Lin J, Epel E, Cheon J, Kroenke C, Sinclair E, Bigos M,
et al. Analyses and comparisons of telomerase activity and
telomere length in human T and B cells: insights for epide-
miology of telomere maintenance. J Immunol Methods 2010;
352:71–80.
48. Klein N, Sefe D, Mosconi I, Zanchetta M, Castro H, Jacobsen M,
et al. Paediatric European Network for Treatment of AIDS 11
Trial Team. The immunological and virological consequences
of planned treatment interruptions in children with HIV in-
fection. PLoS One 2013; 8:e76582.
49. Anselmi A, Vendrame D, Rampon O, Giaquinto C, Zanchetta
M, De Rossi A. Immune reconstitution in human immunode-
ficiency virus type 1-infected children with different virologi-
cal responses to antiretroviral therapy. Clin Exp Immunol 2007;
150:442–450.
50. Freguja R, Gianesin K, Mosconi I, Zanchetta M, Carmona F,
Rampon O, et al. Regulatory T cells and chronic immune
activation in human immunodeficiency virus 1 (HIV-1)-
infected children. Clin Exp Immunol 2011; 164:373–
380.